This invention relates to a stable liquid thromboplastin reagent with a long shelf-life and a method of producing it.
The operation of the coagulation and fibrinolysis pathways of the blood system can be tested at many stages for abnormalities. One of the most commonly used tests is the prothrombin time test (PT). A sample of blood or plasma is added to thromboplastin in the presence of calcium, and the time needed to form a clot is measured. Factor VII is activated by the thromboplastin, which through factors V and X, causes the formation of thrombin from prothrombin (Factor II). The thrombin formed cleaves fibrinogen to insoluble fibrin. The time measured from the mixing of thromboplastin and calcium with a blood sample to the formation of a clot is a measure of the concentration or activity of the coagulation factors involved. This test is used to monitor oral anticoagulant therapy in order to insure that the proper amount of anticoagulant is given the patient. It is also used to test the performance of the coagulation system.
Thromboplastin is the primary reagent for the above tests. Currently, it is obtained from mammalian tissue, usually rabbit brains. Other thromboplastin-rich tissue, such as human brain, human placenta and bovine brain can be used, but is has been found that for cost, performance and availability, rabbit brain tissue is a suitable source of thromboplastin.
The sensitivity of a thromboplastin reagent rests on a number of factors, such as the final reagent composition, which may include buffers, salts and stabilizers; the method of extracting the thromboplastin from tissue; and the original source of the tissue. Most of the prepared thromboplastin reagents on the market today are only available as lyophilized materials, primarily for reasons of reagent stability. Reconstituted lyophilized thromboplastin reagent has a shelf life of approximately four days.
Stability of this reagent is important to the clinician, or user, as it is expensive and the longer the shelf life, both of the opened and unopened reagent container, the less reagent that must be discarded due to expired shelf-life time.
There are inherent problems associated with a lyophilized product that are either reduced or eliminated in a liquid product. These include (1) variability in the filling of the vials before lyophilization; (2) shelf-to-shelf, and shelf positional differences in the lyophilization cycle (freezing and heating); (3) pipette errors associated with reconstitution and/or wrong volume additions when reconstituting the powder; and (4) water used to reconstitute may not be pure and/or may be contaminated with microorganisms.
A lyophilized reagent is inherently more turbid than a liquid reagent. Reducing turbidity of the reagent is also an important factor in producing a better reagent as the clot must be detected as soon as it forms in the PT test.
There are many ways to extract thromboplastin from tissues. The most common is to extract the thromboplastin-rich tissue in water or saline solutions at 25.degree.-50.degree. C. A saline-tartrate solution can be used, after which the extract is centrifuged to remove large particles. The supernatant contains the active thromboplastin along with sodium chloride and sodium tartrate (U.S. Pat. No. 3,522,148). This extract can be further processed. For example, calcium lactate, glycine, carboxymethylcellulose and imidazole can be added to the thromboplastin extract. Each additive has an effect on the sensitivity of the reagent. In general, an acceptable thromboplastin reagent must produce a PT of 9-15 seconds with a normal blood sample.
There are also a number of other processes used to manufacture more sensitive rabbit brain thromboplastin. Hawkins et al., in WO 90/05740, published on May 31, 1990, disclose a method of extracting thromboplastin from tissue using barium sulfate, chaotropic agents and nonionic detergents. However, the process produces only a lyophilized thromboplastin reagent and not a liquid one.
Another patent application, DE 3150594A1, discloses a similar process. Rabbit brain powder is mixed with cellulose powder and washed with sodium acetate buffer at Ph 6.5-8 to remove contaminants. It is then extracted with surfactants in the presence of calcium ions. Again, the thromboplastin produced is stable only in lyophilized form, with a short shelf life once it has been reconstituted.
A liquid thromboplastin is currently available from Pacific Hemostasis, Inc. Although it would appear that this reagent has overcome some problems associated with lyophilized reagents, it is not available as a single vial reagent. Two solutions, in separate vials must be combined to yield a reagent with only a one month stability.
Therefore, in terms of convenience, stability and reliability, a liquid thromboplastin reagent would be of value to the clinical and research laboratories.